Role of DNA methylation in cocaine addiction Abstract Drug addiction is a chronic, relapsing brain disorder characterized by compulsive drug seeking and use despite harmful consequences. It is an urgent social and health problem contributing to more than 90,000 deaths and incurs a yearly cost of over $700 billion in the United States (see NIDA website). It is believed that long-term maladaptive changes in the mesolimbic dopamine reward system play a central role in the development of addictive disorders. However, the underlying molecular mechanism remains largely unknown. The long-lasting effect of drugs on animal behavior and the risk of relapse in human addicts indicate that some stable changes in the brain reward system induced by drugs of abuse mediate these long-term behavioral adaptions. Accordingly, accumulating evidence suggests that drug-induced epigenetic changes, particularly DNA methylation and histone modifications changes, play important roles in drug addiction. However, due to technical difficulties in dealing with the cellular heterogeneity of the mammalian brain, most of the molecular studies performed so far used mixed cell populations, making interpretation of the available data difficult. Consequently, limited progress has been made in understanding the molecular basis of drug addiction. To overcome the issue of cell heterogeneity and to advance our understanding of the epigenetic mechanisms underlying drug addiction, we propose to comprehensively analyze the role of DNA methylation in drug addiction in a neuron subtype- and projection-specific manner using a clinically relevant intravenous cocaine self-administration (IVSA) model. Specifically, we seek to elucidate how DNA methylation in ventral tegmental area (VTA) dopaminergic neurons regulates cocaine reinforcement. To achieve this goal, we have established the following specific aims: 1) Profile transcriptome and methylome of VTA dopaminergic (DA) neurons using a mouse cocaine IVSA model; 2) Functional analysis of key genes regulating DNA methylation in VTA DA neurons; 3) Understand the role of DNA methylation in cocaine reinforcement in projection-specific VTA DA neurons. Completion of the proposed study will not only advance our understanding of how DNA methylation contributes to drug addiction, but also reveal novel therapeutic targets for treating this disorder. Importantly, our study provides a novel, broadly applicable strategy for understanding epigenetic regulation in a neuron subtype- and projection-specific manner.